Water is one of the major if not the predominant, components of many food products. Consequently, changes in the physical state of water in food systems and/or the extent of its interactions with other food components during storage cause structural and textural changes in foods, which, in some cases, are detrimental to their quality. This is particularly a problem in frozen foods, such as meat, fish, desserts, frozen dough products, and frozen fruits and vegetables (Zhu et al., J. Food Eng. 66:69-76 (2005); Regand et al., J. Dairy Sci. 85:2722-2732 (2002); Hartel, R. W. Solid-liquid equilibrium: crystallization in foods. In Physical Chemistry of Foods (Schwartzberg, H. G., Hartel, R. W., eds.) Marcel-Dekker, Inc.: New York; 1992, p. 47; Hagiwara et al., J. Dairy Sci. 79:735-744 (1996); Arbuckle, W. S. Ice Cream. 4th ed., Van Nostrand Reinhold, New York, N.Y., 1986).
During freezing, which is a process of ice crystallization from super-cooled water, first ice nucleation occurs, followed by ice re-crystallization (Mutaftschiev, B. In Handbook of Crystal Growth, Vol. 1, Hurle, D. T. J., Eds., North Holland Elsevier Science Publishers, Amsterdam, The Netherlands, 1993; pp. 189-247). The size distribution of ice crystals formed during this re-crystallization stage has a strong influence on the texture of frozen foods (Regand et al., J. Dairy Sci. 85:2722-2732 (2002); Hartel, R. W. Solid-liquid equilibrium: crystallization in foods. In Physical Chemistry of Foods (Schwartzberg, H. G., Hartel, R. W., eds.) Marcel-Dekker, Inc.: New York; 1992, p. 47) and structural integrity of cell membranes (DeVries, A. L. Survival at freezing temperatures. In Biochemical and Biophysical Perspectives in Marine Biology, Sargent, J. and Mallins, D. W., Eds., Academic Press, London, 1974, pp. 289-330; Beall, P. Cryobiology 20:324-224 (1983)).
For instance, although ice crystals in the range of 15-20 μm bestow a desirable smooth texture to ice cream, those that are larger than 40 μm impart an unacceptable coarse and grainy texture to ice cream (Hagiwara et al., J. Dairy Sci. 79:735-744 (1996); Arbuckle, W. S. Ice Cream. 4th ed., Van Nostrand Reinhold, New York, N.Y., 1986). Water separation in the form of re-crystallized ice in frozen dough-type products (e.g., frozen pizza, and bread rolls) alters macromolecular interactions in the dough network structure, which results in poor textural quality at the time of baking and consumption.
Temperature fluctuations during storage and handling of frozen foods promote ice crystal growth. The crystal growth rate is very slow at lower storage temperatures, especially when the product is stored below its glass transition temperature (Levine et al., Cryo-Lett. 9:21-63 (1988); Simatos et al., Cryo.-Lett. 10:77-84 (1989); and Slade et al., Crit. Rev. Food Sci. Nutr. 30:115-360 (1991)). For ice cream, the glass transition temperature is typically in the range of −30 to −40° C., depending on the sugar ingredient used (Levine et al., Agric. Food Chem. 1:315-396 (1989)). Above the glass transition temperature, the greater molecular mobility of water leads to faster growth of ice crystals. Because the typical average storage temperature in household freezers is well above −20° C. and fluctuates because of automatic defrost cycles (Miller-Livney et al., J. Dairy Sci. 80:447-456 (1997)), formation of large ice crystals and deterioration of textural qualities of frozen foods is a common occurrence under household conditions (Fennema, O. Food Aust. 45:374 (1993)). Thus, one of the major challenges faced by frozen foods manufacturers is developing appropriate technological conditions and ingredient formulations that can inhibit ice crystal growth during storage and handling.
Previously, it has been found that the addition of hydrocolloids, such as gums and polysaccharides, to frozen foods retards the rate of ice crystal growth (Cornwell, A. S. Adv. Chem. Ser. Amer. Soc. 25:59-70 (1960)). The reduced rate of ice crystal growth has been attributed to increased viscosity of the serum phase, which slows down molecular mobility of water (Blond, G. Cryobiology 25:61-66 (1988); Budiaman et al., J. Dairy Sci. 70:547-554 (1987); Harper et al., J. Food Sci. 48:1801-1809 (1983); Flores et al., J. Dairy Sci. 82:1399-1407 (1999)). The reduced rate of ice crystal growth has also been attributed to a possible increase of the glass transition temperature (Hagiwara et al., J. Dairy Sci. 79:735-744 (1996)). Available evidence indicates that hydrocolloids have no or only a marginal effect on heterogeneous nucleation temperature of supercooled water, but they have a measurable effect on ice crystal growth (Flores et al., J. Dairy Sci. 82:1399-1407 (1999)). However, there is no consensus on the mechanism because results from various studies have been contradictory (Flores et al. J. Dairy Sci. 82:1399-1407 (1999); Buyong et al., J. Dairy Sci. 71:2630-2639 (1988); Goff et al., J. Dairy Sci. 76:1268-1275 (1993); Muhr et al., J. Food technol. 21:683-689 (1986)).
Several proteins that inhibit ice nucleation have been found in microorganisms (Holt, C. B. Cryo-Lett. 24:269-274 (2003); Gilbert et al., Microbiology 150:171-180 (2004)), fungi (Hoshino et al., Can. J. Bot. 81:1175-1181(2003)), plants (Sidebottom et al., Nature 406:256 (2000); Worrall et al., Science 282:115-117 (1998)), insects (Graham et al., Science 310:461 (2005); Graether et al., Nature 406:325-328 (2000)), and fish species (Yeh et al., Chem. Rev. 96:601-617 (1996); Chen et al., Biophys. J. 77:1602-1608 (1999)).
These antifreeze proteins (AFP), also known as ice structuring proteins (ISP), are polypeptides belonging to structurally diverse families of genetically coded proteins. The fish AFPs are α-helix type, whereas the structure of AFP of cold-tolerant beetles (and other insects) is made of left-handed parallel β-helix containing 15 residues per coil and that of snow fleas is made up of six antiparallel left-handed poly-proline type II helices stacked in two sets of three to form a compact structure with a hydrophilic and hydrophobic face. Regardless of their structural diversity, antifreeze proteins from fish, insects, and plants typically contain a rigid flat ice binding face (Liou et al., Nature 406:322-324 (2000); Graether et al., Eur. J. Biochem. 271:3285-3296 (2004)). This flat face typically contains side chain hydroxyl groups positioned in a two dimensional array that mimics the spacing of oxygen atoms in the hexagonal ice lattice (Pentelute et al., J. Amer. Chem. Soc. 130:9702-9707 (2008); Liou et al., Nature 406:322-324 (2000); Wen et al., Biophys. J. 63:1659-1662 (1992)). This lattice matching is thought to be fundamental to their ice binding function. Most of the AFPs preferentially bind to the prism faces of ice crystals and inhibit their growth.
The antifreeze activity of AFPs from different sources differs in their effectiveness: Whereas fish AFPs typically depress the freezing point of water by as much as 1° C. (Duman et al., Biol. 2:131-182 (1993)), insect AFPs depress the freezing point by more than 5° C. (Graham et al., Science 310:461 (2005); Graham et al., Nature 388:727-728 (1997)) in a non-colligative manner. However, AFPs do not alter the melting point of ice, which remains at 0° C. The difference between the melting and the freezing temperatures in the presence of these proteins is known as ‘thermal hysteresis’, and the existence of this thermal hysteresis (TH) is a direct indication of involvement of a non-colligative mechanism (Kristiansen et al., Cryobiology 51:262-280 (2005)). In addition to depressing the freezing point, AFPs inhibit ice recrystallization. However, it has been observed that some plant-derived AFPs that show ice recrystallization inhibition (RI) do not exhibit thermal hysteresis (Sidebottom et al., Nature 406:256 (2000); Worrall et al., Science 282:115-117 (1998)), i.e., they do not necessarily depress the freezing point. From the standpoint of applications in frozen foods and survival function in organisms, the ability to inhibit ice recrystallization, rather than the thermal hysteresis (i.e., depression of freezing point), is the most important desirable function of AFPs (Sidebottom et al., Nature 406:256 (2000)).
According to Quick Frozen Foods International (October 2006), the total frozen foods market in the United States was $90.3 billion in 2006, of which the market for ice cream and frozen dessert, fruits, and toppings was about $6.86 billion and that of frozen dough and pizza products was about $6.0 billion. Thus, the frozen foods industry constitutes a significant portion of the total food industry in this country. Ice recrystallization in frozen foods affects the quality of these products. For instance, ice crystals in foods eaten in the frozen state impair their sensory attributes and in frozen dough products (e.g., pizza and other prepared frozen foods) it causes toughening and staling, resulting in poor quality during baking. Because of such ice recrystallization-induced quality changes during frozen storage, each year millions of pounds of frozen products are discarded at the retail and consumer level. The frozen foods industry is faced with the challenge of finding a simple way to inhibit ice recrystallization in frozen food products.
Although antifreeze proteins from fish, plants, and insects that thrive in sub-zero temperature conditions can be potentially used to inhibit ice recrystallization in frozen foods, this is not practical for several reasons: The commercial availability of AFPs is limited and therefore the use of AFPs in frozen foods is not cost-effective. Furthermore, the AFPs are unstable at high temperatures and therefore cannot withstand the blanching operation typically used in frozen fruits and vegetables and pasteurization operation normally used in ice cream manufacture. Therefore, there is a need to develop new antifreeze agents and/or ice recrystallization inhibitors to improve food quality of frozen desserts, dough (e.g., frozen pizza), and fruits and vegetables.